Method for preparation of a new antifungal antibiotic

ABSTRACT

Polyfungin, an antifungal antibiotic of the tetraene group. It is prepared biosynthetically with the use of a strain of actinomyces Streptomyces noursei var polifungini ATCC 21581 or its mutants and variants under aerobic conditions, in aqueous nutrient modium containing an appropriate source of carbon, nitrogen, vitamins, growth substances, macro- and microelements and buffering substances, at temperatures of 25*-35*C., at pH value of 6-7 during 3-6 days, and then after termination of the biosynthesis process polyfungin is recovered from the mycelium.

United States Patent Kotiuszko et al.

METHOD FOR PREPARATION OF A NEW ANTIFUNGAL ANTIBIOTIC Inventors: Danuta Maria Kotiuszku; Krystyna Maria Wituch; Danuta Janina Siejko; llalina Morawska; Natalia Porowska; Maria Teresa Horodecka; Maria Waclawa Wolkowicz; Marlena Nowacka; Zofia Eliza Makarowska-Plociennik; Leszek Halski, all of Warsaw, Poland Assignee: Instytut Przemyslu Farmaceutycznego, Warsaw, Poland Filed: Aug. 14, 1973 Appl. No.: 388,091

Related US. Application Data Continuation of Ser. No. 69,672, Sept. 4, 1970, abandoned.

Foreign Application Priority Data Sept. 11, 1969 135793 US. Cl 195/80 R Int. Cl Cl2d 9/00 Field of Search 195/80 R June 24, 1975 [56] References Cited UNTTED STATES PATENTS 2,786,781 3/1957 Vandeputte et a1. v. 195/80 2,797,183 6/1957 Hazen et al........... 2,832,719 4/1958 Vandeputte 195/80 Primary Examiner-A. Louis Monacell Assistant Examiner-Robert .l. Warden Attorney, Agent, or Firm-Stevens, Davis, Miller & Mosher [57] ABSTRACT 5 Claims, 6 Drawing Figures PATENTEI] JUN 24 ms SHEET PATENTEDJUN 24 ms SHEET 336 346 352 ass ae Cellnumber Polgfungin A Fig.3

.4 2085 qaqaw Fig.4

PATENTEDJUN 24 I975 SHEET 0 0 0 0 0 0 a 7. 6 5 4 3 w m 0 Fig.5

Units} ml.

Distribulion curve "Theoretical curve 332 340 348 356 364 3?2 380 383 396 Cell-number 300 ads 316 324 Fig.6

METHOD FOR PREPARATION OF A NEW ANTIFUNGAL ANTIBIOTIC This is a continuation of application Ser. No. 69,672, filed Sept. 4, 1970, now abandoned.

The object of this invention is a method for preparation of a new antifungal antibiotic, called polifurngin, by biosynthesis and with the use of a strain of actinomyces Streptomyces noursei var. polifungini ATCC 21581 or its mutants and variants.

Polifungin is an antibiotic complex in which the presence of at least three biologically active components have been established. It was demonstrated, basing on the results of chemical analysis that the individual components have in the macrolide ring a system of four conjugated double bonds as well as a system of two conjugated double bonds. Each of the three antibiotics entering in the composition of polifungin has also glucosidally-bound mycosamin, a derivative of mannose. Basing on these properties polifungin was included to polyene antibiotics from the tetraene group.

It is known from the literature that nonhomogeneous antibiotics in the group of tetraene antibiotics from which two by two biologically active components have been isolated, are: nystatin/Shenin D. Yu., Kotienko T. V., Ekzempliarow O. N., Antibiotics, 1968, XI", No 5, 387; Shenin D. Yu., Kotienko T. V., Ugliewa M. F., Ekzempliarow O. N., Materialy V Naucznoj Konfierencji Leningradskogo Nauczno-lssliedowatielnogo lnstituta Antibiotikow, 1967, 206/, antimycoin/C. P. Schaffner, l. D. Steinman, R. S. Safferman, H. Lechevalier, Antibiotics Annual 1957/58, 869/ and tetrin/K. L. von Rinehart jr., V. F. German, W. P. Tucker, D. Gottlieb, Justus Liebigs Annalen der Chemie, 1963, 668, 77/.

A comparative distribution of polifungin and nystatin by the method of countercurrent extraction in the Craig apparatus has demonstrated that fraction A of the polifungin complex and nystatin show identical position of the extraction maximum. Fraction B of polifungin has no equivalent in the preparation of nystatin. After carrying out of further comparative studies on the polifungin preparation from fraction A and nystatin it has been demonstrated that polyfungins A, and A exhibit identical position of the extraction maximum as the A and A components of nystatin. The presence of polifungin B in the polyfungin complex differentiates substantially the two antibiotics: nystatin and polifungm.

The second of the described tetraene antibiotics containing more than one biologically active component is tetrin which has been distributed into two components: A and B. Contrary to polifungin, for both components of tetrin, lack of diene group and lower molecular weight was stated, what makes a substantial differentiation between tetrin and polifungin.

The third of the described antifungal antibiotics of this kind is antimycoin. During its chromatographic distribution the presence of two biologically active components have been demonstrated, called antimycoin A and B. The strain producing this antibiotic has lost with the time its function of synthetising component B and it is possible, therefore, to perform comparative studies, wheras literature data concerning this subject are insufficient.

Polifungin B does not correspond to any of the hitherto described tetraene antibiotics what is proved by the difference in the content of carbon atoms in the molecule, above all. Confrontation of the total formulas of the known tetraene antibiotics and of polifungin B is given in table I.

Table l Confrontation of the total formulas of the known tetraene antibiotics and of polifungin A and B Name of antibiotic Total formula Protocidin fll -lb lil Primaricin zu w u Tetrin B CH-HHHXI-MINOH Tetrin A m sksa in PA- I 66 na u H Etruscomycin m n H Rimocidin Cm au ln Amphotericin A 44 n |1 Nystatin /A, and A,/ C H No Polifungin A IA, and A,/ C H No Polifungin B u-ai u-n tn It is evident from the above given arguments that polifungin is at present the only known antibiotic from the tetraene group containing three antifungal tetraenes, namely polifungin A A and 8, what secures a strong antifungal activity of the polifungin complex in vitro both in relation to the yeastlike fungi and to the filamentous fungi, as related in table 2.

Table 2 Antifungal spectrum of polifungin No Name of test strain Polifungin concentration in mcg/ml l3. 14. 15. l6. l7. l8. 19. 20. 21. 22. 23. 24. 25.

++|+| l 1 l 1 l ++++l l+++++l+l+++ll+l The strains studied were isolated from the cases of clinical mycoses. Spectrum was determined by the dilution method in the Sahnuraud substrate and for the strains of Candida albieans additionally by the filamentous test.

no growth of the test strain poor grow th of the test strain good growth of the test strain Table 2a Table 3 Antil'ungal spectrum of polifungin No Name of test strain l. Ahisidia archidis 2.0 50 5.(] 50 2. Aspergillus niger 1.0 1.5 2.0 3.0 3. Aspergillus terreus 56-8 5.0 5,0 50 4. Brettanomyces bruxollensis R-35 0.2 0.3 5. Candida albicans 102 1 1.5 2.0 2.0 6. Candida tropicalis d/l 0,6 0.8 1,0 2.0 7. Cephalosporium acremonium CMl 49137 0.6 1.0 8. Cryptococcus neoformans R-32 0.4 0.6 0.8 2.0 9. Fusarium caucasicum 0.6 0.8 1.5 10. Fusariurn maniliforme var minus 0.8 1.0 3.0 l l. Kloeckera apiculata R-29 0.1 0.2 0,3 0.3 12. Lipomyces starkeyi R 28 0.2 0.4 13. Penicillium chrysogenum -176 3.0. 4.0 5.0 14. Rhizopus arrhizus 0,8 1,0 3.0 15. Rhodotorula flava R14 0.6 0.8 1.5 16. Saccharomyces cerevisiae ATCC 9763 0.4 0.6 0,8 1.0 17. Sporobolornyces salmonicolor R-41 0.4 0.6 0.8 1.0 18. Sporotrichum schenkii 4.0 5.0 5.0 19. Torula sp. "/31 0.4 0.6 1.0 1.5 20. Torulopsis utilis 11/38 I 0.4 0.6 1.0 1.5 21. Trichophyton plicatile G 1.0 1,5 22. Trichithecium roseurn 0.6 1.0 23. Trichosporon sericeum 11-33 2.0 3.0 5.0 5,0 24. Trigonopsis variabilis R-34 0,2 0.3 0.6

Notice: Determinations were made by the method of dilutions in peptoncycast agar with meat extract of pH 6.5.

From the hitherto described above 30 tetraene antibiotics, because of their high toxicity in vivo only two have found clinical application. namely nystatin and pimaricin. Polyfungin promises broader clinical application as antimycosidal medicine in comparison with the above mentioned antibiotics, particularly in relation to saprophytic fungi which are a petential agent for the systemic mycoses as well as it can be also a medicine of great value in infections caused by a fungus from the group of dermatophytes, namely Epidermophyton floccosum. At the same time the polyfungin complex is an antibiotic of low toxicity. In the tests on mice giving per os quantities of grams per kilogram of body weight no toxic action was observed. At intraperitoneal administration the LD,,,, was [000 mg per kilogram of body weight.

The strain of actinomyces producing polifungin was isolated from soil in China. In the taxonomic studies performed according to the recommendation of the Subcommittee for the Problems of the Actinomyees Taxonomy/Intern. Jour. System. Bacter. /l966/l6,313/ and related in tables 3-6 this microorganism showed according to the key for classification of actinomyces, S. A. Waksman The Actinomycets". 1961. vol. 2, p. 156. the essential taxonomic features characteristic for the species of Streptomyces noursei but because of substantial difference in the quality of produced polyene antibiotics it was recognised as a variety of this species and it was called Streptomyces noursei var. polifungini.

The strain of actinomyces Streptomyces noursei var. polifungini has been deposited in the international collection of ATCC 21581 under number Cultural characteristics of the strain Streptomyces noursei var. polifungini Sporu Aerial Substrate Soluble Medium Growth lation mycelium mycclium pigment color" color malt extract Pale purple yeast abounahoun- [227/ CHM 0c 6r none extract dant dant code 7 la agar l2) Seria GY Light grayish Oatmeal aboungood reddish brown 0c 6t none agar [3/ dant /45/ CHM code 5 fe Seria GY lnorganic Light brownish salts-starch good good gray [63/ agar [4/ CHM code 3 fe Coo 6b none Seria GY Glycerol- Light grayish asparagine good weak reddish brown 0c 6t none agar /5/ I45/CHM code 5 fe Seria (3! Color determinations made after 14 days of incubation in the temperature of 211Tv according to E. B. Shirling. D. (iotlib Methods for characterisation of Streptomyces species. intern. Journal of Systematic Bacteriology. 1966. I6. 3l3.

according to H. I). Trcsncr. E. J. Backus. Appl. Mikrobiol. 1963. ll.4.335; Color Harmony Manual. Container Corporation of America. Chicago. Illinois 60603. U.S.A.

" according to H. Prauser. Z. Allg. Mikrobiologie. 1964. 4.1.95; fodc for dctcrminating the colours of aerial mycclia of streptomycetes. composed by H. Frauser from colour of Baumann Farhtonkarte Atlas 1.

Table 4 The growth of actinomyces Streptomyces noursei var. polifingini ATCC 21587 on the medium with different sources of carbon Source of carbon Growth of actinomyces none D-glucose Larabinose Sucrose D'xylose l-inositol D-mannitol D-fructose Ramnose liiil IHII Notice:

-H1lenotes ahoundant growth good growth zweak growth no growth Table 5 The physiological properties of Streptomyces noursei var. poligungini ATCC 21581 Feature studied Results 1. Peptonisation of milk coagulates. then intensively peptonizes and alkalizes 7. Production of hydrogen sulfide does not produce 8. Production at anti- Table 5 -Continued The physiological properties of Streptomyces noursei var. poligungini ATCC 21581 Feature studied Results xlantlbiotic from the cycloheximide group, 2 antibiotics acting on the Gram-positive bacteria; polyfungin A A and B. antifungal antibiotic from the pol ene group contuinin system of t ree conjugated doubFe bonds; antifungal antibiotic from the polyene group containing seven conjugated double bonds blotics Table 6 Morphological characteristics of Streptomyces noursei var. polifungini ATCC 2158] Sporo phores res surface size Media denotes as in table 3.

Method according to this invention for the preparation of a new antifungal antibiotic, polifungin, by biosynthesis, consists in the biosynthesis process performed with the use of a strain of actinomyces Streptomyces noursei var polifungini ATCC 21,53] or its mutants and variants under aerobic conditions, in aqueous nutrient medium containing an appropriate source of carbon, nitrogen, vitamins, growth substances, macroand microelements and buffering substances, at temperature of 2535C, at pH value of 6-7 during 3-6 days, and then after termination of the biosynthesis process the mycelium is filtered off, washed with water and extracted with organic solvents, the mycelium is distributed, comulated extracts are concentrated in vacuo, the precipitated antibiotic is filtered off, washed and dried, and then purified and dried.

The culture of the strain Streptomyces noursei var polifungini is conducted on media containing, as source of carbon, simple and/or complex carbohydrates, preferably glucose, starch, dextrin in the quantity of l-IO per cent by weight.

As source of organic nitrogen, vegetable and animal raw materials are used under the form of vegetable extracts of flour from seeds, hydrolysates of vegetable material, hydrolysates of animal material, preferably corn-steep extract, soya-bean flour, dried yeasts, yeast hydrolysates, peptones, urea, fish meals in the quantity of 0.1-5 per cent by weight.

As source of inorganic nitrogen, salts containing ammonium and/or nitrate ion are used, preferably ammonium chloride, sodium nirtate, potassium nitrate, ammonium nitrate, amonium sulphate, ammonium acetate, ammonium citrate in the quantity of 01-3 per cent by weight.

As source of macroand microelements tap water is used.

As buffering substance, preferably calcium carbonate is used in the quantity of 0.1-3 per cent by weight.

As organic solvents, preferably low C -C aliphatic alcohols or their aqueous solutions. acetic acid, pyridine, weak pyridine bases, formamide, dimethylformamide, carbon tetrachloride, ethylene glycol and/or their mixtures are used.

Extraction is carried out at temperature of 1050C.

Concentration of the polifungin extract is done under reduced pressure at temperature not exceeding 50C.

Purification is done by elution of contaminations with solvents at temperature of l5-60C.

As solvents, low aliphatic C C, alcohols are preferably used, their aqueous solutions and/or aqueous solu tions of alcohols containing inorganic salts, preferably sodium hexametaphosphate, acetone, esters, preferably ethyl acetate, aliphatic halogen-derivatives, preferably ethylene chloride, and aromatic hydrocarbons, preferably benzene and their mixtures.

Distribution of the polyfungin complex by paper chromatography in the system of water-saturated butanol and with biological detection by the use of the strain Saccharomyces cerevisiae as test microorganism is shown in FIG. I; in which point no. I of antibiotics drift denotes the preparation of polifungin complex, point no. 2 the preparation of polifungin A, point no. 3 the preparation of polifungin B, point no. 4 a mixture of the preparations of polifungin A and B.

Distribution of the polifungin complex by the method of thin-layer chromatography on silica gel in the system methanol-chloroform-water/2:2:l/ and with biological detection by the application of the same test microorganism is shown in FIG. 2 in which successive numbers of the points of drift correspond to the preparations as above.

Both chromatographic distributions demonstrated the presence of two components of polifungin: fraction A and fraction B with antifungal activity.

Confirmation of the results obtained by the chr0- matographic method is distribution of polifungin by the method of countercurrent extraction at 400 transfers in the system methanol-chloroform-borate buffer /2:2:l/ at pH value of 8.2 shown in FIG. 3, in which the distribution curve also demonstrates the presence of two components, namely fraction A, the maximum of which corresponds to the test-tube no. 352 and fraction B, the maximum of which is found in the test-tube no. 305. The ultraviolet absorption spectra shown in FIG. 4 and the infrared spectra represented in FIG. 5 have demonstrated that both polifungin A and polifungin B are tetraenes.

A successive distribution of polyfungin A by the method of countercurrent extraction at 400 transfers in the system amyl alcohol-isoamyl-alcohol-citrate buffer /1 2: l 7129/ shown in FIG. 6 results in a further distribution of polifungin A into polyfungin A and polifungin A The preparation obtained from fraction B prooves to be a homogeneous substance and it was called polifungin B.

The below mentioned examples illustrate but do not limit the process of polifungin preparation.

Examples of the method for actinomyces culture EXAMPLE I.

The strain of actinomyces Streptomyces noursei var polifungini is cultured during 14 days at temperature of 28C on the agar stock slant of the medium with potato extract. The seed culture on liquid medium is conducted during 42 hours in conical 500 ml flasks of Erlenmayer containing 50 ml medium of following composition:

Pure glucose Ammonium nitrate Corn-steep extract [50% of dry mass Calcium carbonate Tap water she): QQQQQ up to It The medium in the flasks is sterilized in an autoclave during 30 minutes at temperature of l 17C after previous correction of pH to the value of 6.4. So prepared seed medium is inoculated either with an aqueous suspension of spores collected from the culture on an agar medium, or with a cutting of the culture. The culture is carried out at temperature of 28C with flasks placed on a rotary shaker of 220 revolutions per minute. The production culture on a liquid medium is carried out during 96 hours in conical 500 ml flasks of Erlenmayer containing 30 ml medium of following composition:

Technical glucose 71 Potato starch ll) /r Corn-steep extract [50% of dry mass/ 1.0 Ci Dried yeasts 0.2571 Ammonium sulphate 0.4% Calcium carbonate 0.8% Tap water up to 100.071

The medium in the flasks is sterilized in an autoclave during 30 minutes at temperature of 1 17C after previous correction of pH to the value of 7.0. So prepared productions medium is inoculated with 5 per cent by the medium volume of the culture prepared on the seed medium. The culture is conducted at temperature of 28C with flasks placed on a rotary shaking machine of 220 revolutions per minute. The content of antibiotic is determined by the microbiological cylinder-plate method in the presence of the test strain Torula sp.ll/31/insensible to the action of cycloheximide/in a methanol extract of mycelium of actinomyces Streptomyces noursei var. polifungini. Determinations are performed in the presence of a standard nystatin preparation and results are given in comparative units calculated per mash. For culture conducted as given above fermentation broth is obtained with polifungin content of 6695 units/ml.

EXAMPLE [I The strain of actinomyces Streptomyces noursei var polifungini is cultured on the solid and liquid seed medium, as given in example I. The production culture is conducted on liquid medium in conical 500 ml flasks of Erlenmayer containing 30 ml medium of following composition:

Technical glucose 2.0% Potato starch 2.0% Com-steep extract I50)? of dry mass/ 1.0% Dried yeasts 0.25% Ammonium sulphate 0.4% Calcium carbonate 0.8% Tap water up to I000;

The medium in the flasks is sterilized in an autoclave during 30 minutes at temperature 0d 1 17C after previous correction 0d pH to the value of 7.0. So prepared production medium is inoculated with 5 per cent by the medium volume of the culture prepared on the seed medium, as given in example I. The culture is conducted at temperature of 28C with flasks placed on a rotary shaking machine of 220 revolutions per minute for the period of 96 hours. The determination of the antibiotic content is performed, as given in example I, and fermentation broth is obtained with polifungin content of 9235 units/ml.

EXAMPLE [[I The strain of actinomyces Streptomyces noursei var.- polifungini is cultured on the solid and liquid seed medium, as given in example I. The production culture is conducted on liquid medium in conical 500 ml flasks of Erlenmayer containing 30 ml medium of following composition:

Technical glucose 2.0% Potato starch 4.071 Corn-steep extract [50% of dry mass/ HM Dried yeasts 0.25% Ammonium nitrate 05% Calcium carbonate 0.8% Tap water up to 100.091

The medium in the flasks is sterilized in an autoclave during 30 minutes at temperature of l 17C after previous correction of pH to the value of 7.0. So prepared production medium is inoculated with 5 per cent by the medium volume of the culture prepared on the seed medium, such as in example I. The culture is conducted at temperature of 28C with flasks placed on a rotary shaking machine of 220 revolutions per minute for a period of 96 hours. The determination of the antibiotic contents is performed, as given in example 1, and fermentation broth is obtained with polifungin content of 16569 units/ml.

Examples of distribution and purification of antibiotic from the culture EXAMPLE 1V 5 l. of fermentation broth obtained according to example lll with polifungin content of 16569 units/ml is filtered off. 400 g of obtained moist mycelium is washed with 10 I water and then with 0.4 l acetone. The mycelium is extracted three times, 1 hour each, using for each extraction 1.2 l of methanol. Cumulated extracts are evaporated under reduced pressure at bath temperature of 40C to the volume of 50 ml. The precipitate is filtered off, washed with 50 ml of acetone and dried at room temperature. and then contaminants are extracted at room temperature with twice as large amount [weight/volume] of ethylene chloride. After filtration and drying at room temperature, the precipitate is extracted again with twice as large amount /weight- /volume/ of distilled water, and, after filtration, dried under reduced pressure at temperature of 40C. The obtained product contains 5150 polifungin units per 1 mg, and the overall yield of the process in relation to polifungin contained in the fermentation broth is equal to 56%. LD 60.2 mg/kg.

EXAMPLE V Fermentation broth of activity 16569 units/ml is carried out as in example IV, with the difference that instead of ethylene chloride the same quantity of ethyl acetate is used. The obtained product contains 5300 polifungin units in 1 mg, and the overall yield of the process in relation to polifungin contained in fermentation broth is 57%. LD 61.7 mg/kg.

EXAMPLE V1 5 l of fermentation broth with polifungin content of 16569 units/ml is filtered off. 400 g of obtained moist mycelium is washed with water and acetone, the precipitate is filtered off and washed with acetone, as given in example IV. The precipitate is dried at room temperature. The obtained precipitate is suspended in 50 ml of 30% isopropanol with 1% sodium hexametaphosphate added and is heated during 30 minutes at temperature of 60C, then cooled to room temperature and filtered off. The obtained precipitate is washed with 100 ml of acetone and dried under reduced pressure at temperature of 40C. The obtained product contains 6350 polifungin units in 1 mg, and the overall yield of the process in relation to polifungin contained in the fermentation broth is equal to 61%. LD 700 mg/kg,

EXAMPLE VII polifungin ATCC 21581 comprising cultivating said culture under aerobic conditions in an aqueous nutricnt containing 1-10% by weight of a source of carbon, 0.15% by weight of a source of nitrogen and 0.1-3% by weight of a buffering agent at a temperature of 25-3 5C. under a pH of 67 for a period of 3 to 6 days, filtering off the mycelium so produced, washing said mycelium with water, extracting said mycelium with methanol at a temperature of l0-50C. three times, concentrating the cumulated extracts under a reduced pressure at a temperature not exceeding 50C.. filtering off the precipitate so formed and purifying the precipitate by elution of contaminants with solvents selected from the group consisting of ethylene chloride, ethyl acetate, 30% aqueous isopropanol with 1% sodium hexametaphosphate and 30% aqueous isopropanol at a temperature of l5-60C.

2. The method of claim 1 wherein said source of carbon is selected from the group consisting of simple carbohydrates and complex carbohydrates.

3. The method of claim 1 wherein said source of nitrogen is an organic source selected from the group consisting of corn-steep extract, dried yeasts, soya bean flour, yeast hydrolysates, peptone, urea and fish meal.

4. The method of claim 1 wherein said source of nitrogen is an inorganic nitrogen salt selected from the group consisting of ammonium chloride, sodium nitrate, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium acetate and ammonium citrate.

5. The method of claim 1 wherein said buffering agent is calcium carbonate. 

1. A METHOD FOR THE PREPARATION OF A POLIFUNGIN ANTIBIOTIC COMPLEX OF AT LEAST THREE TETAENES OBTAINED FROM THE MYCELIUM OF A CULTURE OF STREPTOMYCES NOURSEI VAR. POLIFUNGIN ATCC 21581 COMPRISING CULTIVATING SAID CULTURE UNDER AEROBIC CONDITIONS IN AN AQUEOUS NUTRIENT CONTAINING 1-10% BY WEIGHT OF A SOURCE OF CARBON,0.1-5% BY WEIGHT OF A SOURCE OF NITROGEN AND 0.1-3% BY WEIGHT OF A BUFFERING AGENT AT A TEMPERATURE OF 25*-35*C. UNDER A PH OF 6-7 FOR A PERIOD OF 3 TO 6 DAYS, FILTERING OFF THE MYCELIUM SO PRODUCED, WASHING SAID MYCELIUM WITH WATER, EXTRACTING SAID MYCELIUM WITH METHANOL AT A TEMPERATURE OF 10*-50*C. THREE TIMES, CONCENTRATING THE CUMULATED EXTRACTS UNDER A REDUCED PRESSURE AT A TEMPERATURE NOT EXCEEDING 50*C. FILTERING OFF THE PRECIPITATE SO FORMED AND PURIFYING THE PRECIPITATE BY ELUTION OF CONTIMINATS WITH SOLVENTS SELECTED FROM THE GROUP CONSISTING OF ETHYLENE CHLORIDE, ETHYL ACETATE, 30% AQUEOUS ISOPROPANOL WITH 1% SODIUM HEXAMETHOSPHATE AND 30% AQUEOUS ISOPROPANOL AT A TEMPERATURE OF 15*-60*C.
 2. The method of claim 1 wherein said source of carbon is selected from the group consisting of simple carbohydrates and complex carbohydrates.
 3. The method of claim 1 wherein said source of nitrogen is an organic source selected from the group consisting of corn-steep extract, dried yeasts, soya bean flour, yeast hydrolysates, peptone, urea and fish meal.
 4. The method of claim 1 wherein said source of nitrogen is an inorganic nitrogen salt selected from the group consisting of ammonium chloride, sodium nitrate, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium acetate and ammonium citrate.
 5. The method of claim 1 wherein said buffering agent is calcium carbonate. 